Novel Methylated DNA Markers Accurately Discriminate Lynch Syndrome Associated Colorectal Neoplasia

Figures & data

Table 1. Patient and lesion characteristics for discovery and validation phases.

Patient/lesion characteristic	Discovery	Biological validation
	Lynch	Sporadic	Lynch
Normal mucosa:
n	18	35	23
Definite LS	15 (83%)	n/a	13 (57%)
Age, median (IQR)	53 (44.8–62.5)	64 (53.5–71.5)	50 (37.5–57)
Sex, % female	61%	49%	57%
Advanced adenoma:
n	18	38	39
Definite LS	10 (56%)	n/a	26 (67%)
Age, median (IQR)	57 (53.3–66.3)	64.5 (56–76.3)	59 (50.5–69)
Sex, % female	33%	40%	59%
Site, % proximal	55%	76%	51%
Tubular adenoma	18	13	37
Sessile serrated polyp	0	25	2
Adenocarcinoma:
n	17	36	26
Definite LS	12 (71%)	n/a	14 (54%)
Age, median (IQR)	59 (54–71)	68.5 (57.8–80.5)	46.5 (36.5–58.8)
Sex, % female	41%	50%	46%
Site, % proximal	6159%	47%	46%

IQR: Interquartile range; LS: Lynch syndrome; n: Number of patients.

Table 2. Biological validation of methylated DNA marker candidates in independent colorectal tissues from Lynch syndrome and sporadic patients.

Discovery method	MDM	Adenoma			Cancer
		Lynch	Sporadic	p-value	Lynch	Sporadic	p-value
Lynch discovery	PITX1	0.86	0.97	0.034	0.98	1	0.201
	CBLN2	0.86	0.95	0.11	0.97	0.82	0.025
	USP44	0.85	0.96	x0.066	0.96	0.97	0.671
	STK32B	0.79	0.9	0.15	0.95	0.95	0.932
	CNTFR	0.79	0.93	0.031	0.92	0.87	0.378
	ALKBH5	0.68	0.44	0.015	0.89	0.45	<0.001
	ADCY4	0.81	0.75	0.508	0.88	0.93	0.415
	ADM	0.63	0.62	0.872	0.81	0.42	<0.001
	ANTXR1	0.53	0.96	<0.001	0.71	0.79	0.378
Sporadic discovery	ARHGEF4	0.89	0.97	0.127	0.99	0.97	0.554
	SFMBT2_897	0.80	0.96	0.007	0.99	0.9	0.082
	OPLAH	0.99	0.99	0.83	0.97	1	0.416
	ELMO1	0.78	0.85	0.368	0.92	0.84	0.25
	LRRC4	0.86	0.88	0.795	0.92	0.98	0.188
	VAV3	0.73	0.95	0.001	0.87	0.89	0.829
	DAB2IP	0.78	0.89	0.134	0.81	0.87	0.478
	PDGFD	0.72	0.9	0.02	0.79	0.88	0.293
	CHST2_7889	0.68	0.88	0.013	0.77	0.91	0.08
	AK055957	0.62	0.87	0.003	0.62	0.76	0.192
MT-sDNA test	BMP3	0.57	0.86	<0.001	0.77	0.8	0.746
	NDRG4	0.60	0.93	<0.001	0.69	0.9	0.02

MDM: Methylated DNA marker; MT-sDNA test: Multi-target stool DNA test.

Figure 1. Box plot distributions of selected methylated DNA marker candidates in colorectal tissues from Lynch syndrome and sporadic patients from biological validation phase.

(A) MDMs showing similarly high neoplasm discrimination across Lynch syndrome and sporadic patients. (B) MDMs showing relatively higher neoplasm discrimination in Lynch syndrome patients. (C) MDMs showing relatively higher discrimination in sporadic patients.

A: Adenoma; C: Colorectal cancer; MDM: Methylated DNA marker; N: Normal.

Figure 2. Heat matrices: methylation intensity of methylated DNA marker candidates in independent colorectal tissues from biological validation phase.

(A) Lynch syndrome and (B) sporadic tissues. Increasing intensity of yellow-red color spectrum in boxes indicates methylation strand counts in deciles above the 90th percentile values for the control groups (histologically normal mucosa) of each candidate methylated DNA marker (rows) in each tissue sample (columns). Black boxes indicate values falling below the 90th percentile in controls.

Figure 3. Discrimination of selected methylated DNA marker candidates for colorectal neoplasia as assessed by receiver operator curves.

AUC in each graph are shown for OPLAH alone, a panel of novel methylated DNA markers without OPLAH (ARHGEF4, LRRC4, ANTXR1, PITX1) and the combination of BMP3 + NDRG4 with (A) Lynch adenomas, (B) Lynch cancers, (C) sporadic adenomas and (D) sporadic cancers.

AUC: Area under the curve.

Table 3. Functions of genes methylated in Lynch syndrome colorectal cancers.

Gene	Protein	Function	Cancer associations/mechanism^†
ADCY4	Adenylate cyclase 4	Signal transduction	Prostate cancer/hypermethylation
ADM	Adrenomedullin	Signal transduction	CRC, PDAC, renal cell cancer/upregulation
ALKBH5	RNA demethylase ALKBH5	Cell differentiation	Glioblastoma, breast cancer/upregulation
ANTXR1	Anthrax toxin receptor 1	Cell attachment and migration	CRC, lung cancer, breast cancer/upregulation
CBLN2	Cerebellin 2 precursor	Synapse assembly	CRC/hypermethylation
CNTFR	Ciliary neurotrophic factor receptor	Cellular adhesion/signal transduction	Glioma/upregulation
PITX1	Pituitary homeobox 1	Transcriptional regulation	CRC, PDAC, gastric, bronchial, prostate, oral cancers/downregulation
STK32B	Serine/threonine kinase 32B	Signal transduction	Oral squamous cell cancer, breast cancer/upregulation
USP44	Ubiquitin carboxyl-terminal hydrolase 44	Cell cycle regulation	CRC/transcriptional silencing, hypermethylation

† Information on table based on literature review [32–49].

CRC: Colorectal cancer; PDAC: Pancreatic ductal adenocarcinoma.